The goal of this project is to develop the use of RNA enzymes as reagents for the site specific cleavage (a consequent inactivation) of HIV and other high molecular weight RNAs in vitro and in vivo. Recent success in several labs using the hammerhead ribozyme to inactivate a number of RNAs (including HIV sequences) in bacterial, plant and animal cells emphasize the value of this emerging technology. This project will focus on refining and expanding the biochemical description of the cleavage of small and large RNAs by the hammerhead ribozyme. A kinetic description of the cleavage of matched and mismatched targets will be developed with the goal of predicting both the rate and specificity of the reaction. A strategy is proposed to identify sites in large RNAs that are particularly susceptible to cleavage. The inhibition of hammerhead cleavage by antibiotics will be explored. Finally, recently developed methods for in vitro evolution will be used to select for improved versions of the hammerhead and for new ribozymes that cleave pre-defined RNA targets.